The present invention relates to ribozymes and their expression systems.
A hammerhead ribozyme is one of the smallest catalytic RNA Molecules (Kruger et al., 1982; Guerrier-Takada et al., 1983). Because of its small size and potential as an antiviral agent, numerous mechanistic studies (Dahm and Uhlenbech, 1991, Dahm et al., 1993; Eckstein and Lilley, 1996; Pontius et al., 1997; Lott et al., 1998; Zhou et al., 1996, 1997; Zhou and Taira, 1998) and studies directed towards application in vivo have been performed (Erickson and Izant, 1992; Murray, 1992; Rossi, 1995; Eckstein and Lilley, 1996; Prislei et al., 1997; Turner, 1997; Scanlon, 1997). Many successful experiments, aimed at the use of ribozymes for suppression of gene expression in different organisms, have been reported (Sarver et al., 1990; Dropulic et al., 1992; Ojwang et al, 1992; Yu et al, 1993; Zhao and Pick, 1993; Inokuchi et al, 1994; Yamada et al., 1994; Ferbeyre et al, 1996; Fujita et al, 1997; Kawasaki et al, 1998). However, the efficacy of ribozymes in vitro is not necessarily correlated with functional activity in vivo. Some of the reasons for this ineffectiveness in vivo are as follows. i) Cellular proteins may inhibit the binding of the ribozyme to its target RNA or may disrupt the active conformation of the ribozyme. ii) The intracellular concentration of metal ions essential for ribozyme-mediated cleavage might not be sufficient for functional activity. iii) Ribozymes are easily attacked by RNases. However, we are now starting to understand the parameters that determine ribozyme activity in vivo (Bertrand and Rossi, 1996; Bertrand et al., 1997; Gebhard et al., 1997). Studies in vivo have suggested that the following factors are important for the effective ribozyme-mediated inactivation of genes: a high level of ribozyme expression (Yu et al., 1993); the intracellular stability of the ribozyme (Rossi and Sarver, 1990; Eckstein and Lilley, 1996); co-localization of the ribozyme and its target RNA in the same cellular compartment (Sullenger and Cech, 1993; Bertrand et al., 1997); and the cleavage activity of the transcribed ribozyme (Thompson et al., 1995). Recently, it was shown that these various features depend on the expression system that is used (Bertrand et al., 1997).
The RNA polymerase II (pol II) system, which is employed for transcription of mRNAs, and the polymerase III (pol III) system, employed for transcription of small RNAs, such as tRNA and snRNA, have been used as ribozyme expression systems (Turner, 1997). Transcripts driven by the pol II promoter have extra sequences at the 3xe2x80x2 and 5xe2x80x2 ends (for example, an untranslated region, a cap structure, and a polyA tail), in addition to the coding region. These extra sequences are essential for stability in vivo and functional recognition as mRNA. A transcript containing a ribozyme sequence driven by the pol II promoter includes all those sequences, unless such sequences are trimmed after transcription (Taira et al., 1991; Ohkawa et al., 1993). As a result, in some case, the site by which the ribozyme recognizes its target may be masked, for example, by a part of the coding sequence. By contrast, the pol III system is suitable for expression of short RNAs and only very short extra sequences are generated. In addition, expression is at least one order of magnitude higher than that obtained with the pol II system (Cotten and Birnstiel, 1989). Thus, it was suggested that the pol III system might be very useful for expression of ribozymes (Yu et al., 1993; Perriman et al., 1995). However, in many cases, the expected effects of ribozymes could not be achieved in spite of the apparently desirable features of the pol III system (Ilves et al., 1996; Bertrand et al., 1997).
In order to investigate the parameters that determine ribozyme activity in vivo, we designed three types of ribozyme with an identical ribozyme sequence, driven by tRNAVal promoter which is a pol III promoter, and demonstrated that the entire structure of the transcript (ribozyme to which the sequence of tRNAVal is added (hereinafter termed xe2x80x9ctRNAVal-ribozymexe2x80x9d)) determined not only cleavage activity but also the intracellular half-life of the ribozyme. All the chimeric tRNAVal-ribozymes that were transcribed in the cell nucleus were exported to the cytoplasm. Thus, the ribozymes and their target were present within the same cellular compartment. Under these conditions, we found that the intracellular half-life and the steady-state level of each tRNAVal-ribozyme were the major determinants of functional activity in vivo. Moreover, we demonstrated that cells that expressed a specifically designed ribozyme with the longest half-life in vivo were almost completely resistant to a challenge by HIV-1. Further, by establishing a small bulge structure (xe2x80x9cbulgexe2x80x9d refers to, in the case where RNA adopts a hairpin structure, a portion where there is a protruding single-stranded structure of unmatched base pairs) at the amino-acyl stem portion of the tRNAVal structure, avoidance of recognition from the mature enzyme can be achieved and as a result, any RNA sequence comprising a ribozyme sequence connected to the 3xe2x80x2 end can be made to exist intracellularly in a form where it is connected to tRNAVal. Any RNA comprising a ribozyme sequence connected to the 3xe2x80x2 end of the tRNAVal structure of the present invention, due to the properties of the tRNA structure, is transported stably and efficiently to the cytoplasm. This is of particular importance for the intracellular function of the ribozyme.
A summary of the present invention is presented as follows:
1. A ribozyme comprising a nucleotide sequence having the following base sequence (I) or (II):
base sequence (II) (SEQ ID NO. 1): 5xe2x80x2-ACCGUUGGUUUCCGUAGUGU AGUGGUUAUCACGUUCGCCUAACACGCGAAAGGUCCCCGGUUCGAAACCGGGCAC UACAAACACMCACUGAUGAGGACCGAAAGGUCCGAAACGGGCACGUCGGAAACGG UUUU[[U]]-3xe2x80x2
base sequence (II)(SEQ ID NO. 2): 5xe2x80x2-ACCGUUGGUUUCCGUAGUGUAGUG GUUAUCACGUUCGCCUAACACGCGAAAGGUCCCCGGUUCGAAACCGGGCACUACM ACCMCACACMCACUGAUGAGGACCGAAAGGUCCGAAACGGGCACGUCGGAAACG GUUUU[[U]]-3xe2x80x2.
2. An expression vector comprising DNA encoding the ribozyme according to 1 above.
3. A method of producing the ribozyme according to 1 above comprising transcribing to RNA with expression vector DNA as a template, wherein said expression vector DNA comprises DNA encoding the ribozyme according to 1 above.
4. A pharmaceutical composition comprising the ribozyme according to 1 above or the expression vector according to 2 above, as an effective ingredient.
5. The pharmaceutical composition according to 4 above for the prevention and/or treatment of acquired immune deficiency syndrome.
6. A method of specifically cleaving a target RNA using the ribozyme according to 1 above.
7. The method of 6 above wherein the target RNA is HIV-1 RNA.
8. An RNA variant (mature tRNAVal) adopting the following secondary structure (I), wherein said RNA variant comprises a bulge structure introduced in the region in which hydrogen bonds form between nucleotides 8 to 14 and nucleotides 73 to 79. 
9. The RNA variant of 8 above wherein a bulge structure is introduced by substituting all or part of the sequence of the region corresponding to nucleotides 73 to 79 within a nucleotide sequence of an RNA adopting secondary structure (I).
10. The RNA variant according to 8 above consisting of the sequence of a region corresponding to nucleotides 1-80 within a nucleotide sequence represented by SEQ ID NO: 1.
11. The RNA variant according to 8 above consisting of the sequence of a region corresponding to nucleotides 1-86 within a nucleotide sequence represented by SEQ ID NO: 2.
12. An RNA comprising the RNA variant of 8 above and a selected RNA chain linked thereto.
13. The RNA according to 12 above wherein selected RNA chain is a ribozyme or an antisense RNA.
14. The RNA according to 12 above wherein a bulge structure is formed with any nucleotide of an RNA chain linked to the 3xe2x80x2 terminus and any nucleotide of the region of nucleotides 8 to 14 within the nucleotide sequence of an RNA adopting secondary structure (I).
15. An expression vector comprising DNA encoding the RNA of 12 above.
Having consideration for the transcription amount, stability and post-transcription activity of ribozymes, we selected human tRNAVal promoter which is involved in a polymerase III system, as an expression system therefor, and examined whether there was any difference in ribozyme effect in vivo due to the way in which the ribozyme was linked to this promoter. In other words, we focussed on intracellular stability which is an important factor in obtaining significant ribozyme effect in vivo, and post-transcription activity, and set out to clarify the relationship between the high-order structure of ribozymes and these factors.
First, we designed a hammerhead ribozyme targeting a relatively conserved sequence of HIV-1, and constructed four expression systems by attaching this gene to downstream of the tRNAVal promoter via various sequences. As a vector for the construction of these expression systems we used pUC19 (Takara), however, other vectors such as pGREEN LANTERN (Life Technologies Oriental, Inc.) and pHaMDR (HUMAN GENE THERAPY 6:905-915 (July 1995)) may also be used. Also, oligonucleotide sequences necessary for the construction of these expression systems can be chemically synthesized with a DNA/RNA synthesizer (Model 394; Applied Biosystems, Division of Perkin Elmer Co. (ABI), Foster City, Calif.).
From predictions made using Zuker""s method, it was thought that differences in the linker sequence used to connect the tRNAVal promoter and hammerhead ribozyme would exert great influence on the secondary structure of the recognition site of the ribozyme (See FIG. 1). According to this prediction map, it was clear that whereas the overall secondary structure of the ribozyme was almost the same, the degree of freedom at the substrate-binding site differed greatly. It is clear that whereas both substrate binding sites form a stem structure within the molecule in Rz1, one binding site in Rz2, and both binding sites in Rz3 protrude to the outside. In the case of Rz3, the protruding substrate binding site may be masked by protein. However, since a ribozyme is an RNA enzyme and both binding ability and disassociation ability with a substrate are important factors in its activity. Rz3 was expected to be the best in terms of cleavage ability. We performed a reaction using intracellularly transcribed ribozymes, in an in vitro system under the following conditions: 40 mM Tris-Cl (pH8.0), 8 mM MgCl2, 5 mM DTT, 2 mM Spermidine, 2 U/xcexcl RNase inhibitor, 30 xcexcg total RNA. At this time, the ribozyme content in total RNA was made constant. The results showed that ribozyme activity toward short substrates that were transcribed in vitro and radioactively labeled depended on the degree of freedom at the recognition site (See FIG. 2). Further, stability of the ribozymes was examined, with the expression amount of a control gene made constant, we conducted a comparative study of each ribozyme amount. The difference in ribozyme structure also affected stability. The reason why structures having such little overall difference exert this influence is not clear, however, a difference of approximately 25 times was exhibited as between the most stable and the least (FIG. 3B).
We next examined the relationship between ribozyme activity in vitro and in vivo effect, which as discussed above is as yet unclear. First, using a luciferase gene as a reporter gene, a system for evaluating ribozyme effect was constructed wherein ribozymes are allowed to act on a fusion gene of this reporter gene and the sequence pNL4-3 (an HIV-1 clone), and luciferase activity in the cell extract is measured (See FIG. 3A). A comparison of each of the ribozymes showed that the one with the highest intracellular stability exhibited highest activity suggesting the importance of stability (See FIG. 4).
The above discussion relates to an evaluation of ribozyme effect on an artificial fusion gene of a luciferase gene and a HIV-1 sequence, in cultured cells. Therefore, it may be difficult to judge that these results are the equivalent of results that could be obtained in an organism. Thus, we performed an evaluation of ribozyme effect against actual HIV-1 (See FIG. 5). A ribozyme expression system transformant was infected with HIV-1, and virus growth was measured by measuring the amount of p24 (core protein of the virus) produced in serum. Results indicating a similar trend to our evaluation in cultured cells were obtained. Further, it was clear that the ribozyme with the highest in vitro stability exhibited very high inhibitory effect with production of p24 inhibited by 99% (See FIG. 6C). In contrast, the ribozyme expression system having the highest cleavage activity in vitro was mostly unable to inhibit growth of the virus.
In this manner it became clear that the evaluation of ribozyme effect with the virus indicated the same trend as the evaluation with the artificial substrate in cultured cells. Thus, the results of the once-over evaluation we performed are thought to be a rough guide to the effect of the ribozymes in vivo. Further, from the results of these experiments both in vitro and those dealing with a virus, it is clear that to obtain significant intracellular effect of the ribozyme, while activity is important, intracellular stability is of greater importance. The fact shown in the case described herein, that even ribozymes whose sequences have little differences lead to great differences in effect in vivo depending on the linkage to the expression system, must be fully considered. The above result also suggests that it is important to design ribozymes with high stability while considering the influence of higher structures comprising added sequences for expression on their stability.
tRNA is first recognized intracellularly with its promoter sequence consisting of sequences known as A box and B box within its structural sequence, and transcribed with the extra sequences connected to the 5xe2x80x2 and 3xe2x80x2 ends. Next, with to the action of a plurality of mature enzymes which exist within cells, extra sequence are eliminated to form mature tRNA. In the action of these enzymes, the structure of the portion known as the amino-acyl stem becomes an important determinant of structure recognition. We found that by establishing a small bulge structure at the portion, it was possible to avoid the action of the mature enzyme. For example, in FIG. 1, each of Rz1, Rz2 and Rz3 has a small bulge structure at the amino-acyl stem, and as a result, the ribozyme sequence of the transcribed RNA is not eliminated (FIGS. 3B, 7A and determined base sequence). Such a property is not due to the 3xe2x80x2 end sequence being a ribozyme but is due to the bulge structure at the amino-acyl stem portion, consequently, any RNA sequence comprising antisense may be used.
Typically tRNA undergoes a series of processings before transportation from the nucleus including the removal of extra sequences at the 5xe2x80x2 and 3xe2x80x2 ends by a mature enzyme, removal of any introns by splicing mechanism, modification of specific bases, and addition of a sequence consisting of 5xe2x80x2-CCA-3xe2x80x2 to the 3xe2x80x2 terminus and, in some cases addition of amino acids suitable for said tRNA (amino-acylation).
However, the tRNA of the present invention is actively transported out of the nucleus without being subject to, from among the above series of modifications, at least, removal of extra sequences at the 5xe2x80x2 and 3xe2x80x2 ends, addition of a CCA sequence to the 3xe2x80x2 end, and subsequent amino acylation (FIG. 7A). This is likely because establishing a bulge in the amino acyl stem portion results in avoidance of action by mature enzymes and following addition of CCA sequence followed by amino acylation, and the entire structure resembles that of the original tRNA. This inference was also supported by the fact that the one with degenerated tRNA structure (Rz4), according to structure predictions using computers, was not transported to the cytoplasm. (FIG. 7C). This property of being transported from the nucleus to the cytoplasm does not depend on the ribozyme sequence at the 3xe2x80x2 end, and thus it is thought that any RNA sequence such as antisense RNA may be used similarly.
In recent years, it has come to be understood that where antisense RNA and ribozyme RNA are expressed intracellularly, in order to elicit their function, it is important that their distribution in a cell is within the cytoplasm. Since RNAs of the present invention form stable tRNA-like structures by themselves, they have the function of definitely transporting to the cytoplasm without exerting great influence on the higher-order structure of the RNA linked to the 3xe2x80x2 end (which is an extremely important property when the RNA linked to the 3xe2x80x2 end is a functional RNA such as a ribozyme or antisense RNA). Further, when the RNA is made into DNA, it functions as a promoter irrespective of cell type and it has a broad range of hosts (originally of human derivation, but should be able to express in at least all mammals.) In short, the present invention is most suitable for an antisense RNA and ribozyme RNA expression systems, and can become an important tool for experiments using cultured cells and for gene therapy in the field of medicine. Further, through the technique of molecular evolution engineering, RNA molecules with functions not found in nature, are recently being artificially created. If these molecules exhibit their functions intracellularly, particularly in the cytoplasm, then the present invention will be able to be used as an expression system for these RNA molecules.
Using the tRNAVal-ribozyme of the present invention it is possible to specifically cleave a target RNA, particularly an HIV-1 RNA.
The tRNAVal-ribozyme of the present invention can be used as a medicine especially for the prevention and/or treatment of acquired immune deficiency syndrome. For example, the transcription of HIV can be inhibited by encapsulating the tRNAVal-ribozyme of the present invention in a liposome, administering this to an organism and allowing incorporation into cells comprising HIV. Further, transcription of HIV can be inhibited by incorporating DNA encoding the tRNAVal-ribozyme of the present invention in a vector such as virus, introducing the vector into cell comprising HIV to allow intracellular expression of the vector thereby effecting production of the tRNAVal-ribozyme of the present invention. Administration of the tRNAVal-ribozyme of the present invention will depend on severity of the conditions of the patient and responsiveness of the organism, and may be conducted in appropriate amount, form of administration and frequency, and over a period until the efficacy of prevention and/or treatment can be recognized, or until alleviation of the patient""s condition is achieved.
The present specification incorporates in its entirety the content of the specification and drawings of Japanese Patent Application No. 10-244755, said application forming the basis of the priority claim of this application.